Measuring concentration of analytes in liquid samples using surface-enhanced Raman spectroscopy

ABSTRACT

A hand-held microfluidic testing device is provided that includes a housing having a cartridge receiving port and a cartridge for input to the cartridge receiving port. An optical detection system in the housing is capable of providing an illuminated electric field useful for Raman spectroscopy. The cartridge may have a sample well. The sample well is loaded with a mixture of water containing the analyte, Raman-scattering nanoparticles and a calibration solution. The calibration solution contains an analog of the analyte differing in its Raman response, for example an isotope of the analyte. Optionally, a chemical compound capable of increasing interaction between the analyte and the nanoparticles may be added.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/267,708, filed Sep. 16, 2016, which claims the benefit of U.S.Application Ser. No. 62/219,553, filed on Sep. 16, 2015. U.S. Ser. Nos.15/267,708 and 62/219,553 are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

Parts of this specification relate to research funder under a NationalScience Foundation—SBIR grant, number IIP-1058590, called “Hand-HeldDevice for PPB-level Water Analysis.”

FIELD OF THE INVENTION

The present invention relates to measuring the concentration of analytesin a fluid, for example by using surface-enhanced Raman spectroscopy tomeasure the composition of a trace analyte in an industrial orenvironmental water sample.

BACKGROUND

Currently, the detection and measurement of many analytes at tracelevels within a water sample requires complex laboratory equipment and askilled technician. A variety of laboratory techniques exist. The EPAprovides a list of available and approved techniques for compounds ofconcern. The American Public Health Association, American Water WorksAssociation, and Water Environmental Federation publish Standard Methodsfor the Examination of Water and Wastewater—an extensive treatise onwater analysis methodology. Although a variety of analytical detectionmethods can be used in water analysis, most trace-level analysis fallsupon widely-used and popular, yet complex and expensive, massspectrometry methods.

Raman spectroscopy is a potential alternative for trace analytedetection. Raman spectroscopy provides a chemical signature for acompound (in the form of a unique configuration of peaks in thereflected spectrum), but the Raman signal is typically too weak forpart-per-billion detection. However, the Raman signal can be enhanced bythe presence of an active surface or marker (typically a metal),creating surface-enhanced Raman spectroscopy (SERS). With an adequateinteraction between the analyte and the surface, signal enhancementcreates opportunities to detect very small concentrations. Somecompounds, such as pyridine, naturally interact strongly with gold andsilver surfaces. In some other cases a binding compound may bring theanalyte and metal molecules into sufficiently close contact. Forexample, treatments with octadecylthiol have been used successfully forSERS on planar substrates with some analytes.

In some SERS techniques, the surface is provided by metal nanoparticles.When a metallic nanoparticle smaller than the wavelength of light isintroduced into the sample, the illuminating electric field will createsurface resonances if there are free electrons in the nanoparticle. Thenanoparticles can be gold, silver, or copper beads, for example. Theseoscillating charges create an enhanced local electric field alongcertain directions, which results in a much stronger Raman response.“Hot spot” regions can be created where the SERS signal is greatlyenhanced. These regions are most likely due to nanoparticle alignmentsor aggregates that create even larger electric field enhancements.

U.S. Pat. No. 8,070,956, Hand-Held Microfluidic Testing Device,describes a testing device with a cartridge receiving port for receivinga cartridge. An optical detection system in the housing is disposed toanalyze a sample in channel of the cartridge. In some embodiments,markers such as gold nanoparticles are present in the channel and theoptical detection system is a Raman spectroscopy system. In someembodiments, the channel includes narrow sections along a microfluidicseparation channel that trap gold nanoparticles at a high density. Thisencourages the creation of “hot spots” through nanoparticle density at apredetermined detection location.

Although some researchers have achieved remarkable detection limitsusing SERS, the use of SERS to measure the concentration of a traceanalyte is less developed. In general, Raman signal strength isproportional to the amount of analyte per unit area. However, one issueis that the generation of random nanoparticle hot spots leads to randomsignal enhancements, which interferes with correlating signal strengthto analyte concentration. Another issue is that aqueous sampleconditions other than analyte concentration, for example pH, can alterthe analyte signal strength.

INTRODUCTION

This specification describes the use of surface-enhanced Ramanspectroscopy (SERS) to measure the concentration of various analytes,for example in an industrial or environmental water sample. In at leastsome cases, the composition of trace compounds is measured to within 10%accuracy. A measurement method uses an internal standard in the form ofa known quantity of a reference compound added to a known volume of anaqueous mixture containing the analyte. The reference compound reactssimilarly to the analyte under investigation with the surface butproduces a different Raman spectrum. The reference compound may be, forexample, an isotopologue, isomer, or enantiomer of the analyte or of acompound similar to the analyte. The reference compound is added to themixture in an amount similar to the expected amount of the analyte.

A portable testing device is described having reusable and consumablecomponents. The reusable components include a Raman spectroscopyinstrument having a cartridge holder and software for operating theRaman spectroscopy instrument, optionally stored in a general-purposecomputer. The consumable components include a cartridge, a dispersion ofRaman-scattering nanoparticles and one or more reagents for the analyteunder investigation. The one or more reagents, typically provided insolution, include a reference compound for the analyte and, optionally,one or more chemical compounds capable of increasing interaction betweenthe analyte and the Raman-scattering nanoparticles. The cartridge has acavity adapted to hold a mixture of the analyte, nanoparticles and oneor more reagents in a suitable location in the Raman spectroscopyinstrument. In some embodiments, the device may also include asolid-phase extraction column or other pretreatment devices orchemicals.

In use, a mixture is prepared of a water sample containing the analyteunder investigation, the nanoparticles and the one or more reagents. Atleast some of the mixture is placed on the cartridge. The cartridge isloaded into the Raman spectroscopy instrument. The computer controls theRaman spectroscopy instrument to produce a Raman spectrum of themixture. The produced spectrum includes individual spectra for theanalyte and the reference compound. A ratiometric analysis of theindividual spectra, typically performed by the software, is used tocalculate the concentration of the analyte.

In some cases, the reference standard is an isotopologue of the analyte,or an isotopologue of a compound chemically similar to the analyte. Whenusing the word “isotopologue” we mean to refer (unless clear from thecontext otherwise) to a form of a compound that differs in number ofneutrons from the most common naturally occurring form of that compound.The word “isotope” may also be used for brevity or convenience with thesame meaning even though, strictly speaking, the word isotope should beused with elements rather than molecules. Isotopologues of a compoundhave Raman spectra that are different from the commonly occurring formof the compound. The substitution of deuterium for hydrogen in apyridine molecule, for example, results in a SERS spectrum withessentially the same intensity but with shifted peaks.

In the case of an analyte that is a molecular ion, a chemically similarcompound may be another molecular ion having the same type and number ofelectron acceptors attached by the same type of bonds but to a differentelectron donor. In the case of an analyte with a functional group thatis naturally SERS active under at least some conditions, such as anamine, a chemically similar compound may be a compound with the samefunctional group.

The Raman spectrum generated by the reference standard is produced andrecorded simultaneously with the spectrum generated by the analyte in acomposite spectrum. The Raman intensity of one or more bandscorresponding to a known amount (i.e. concentration) of the referencestandard is used as a reference against which the Raman intensity of oneor more bands corresponding to the analyte is converted into ameasurement of the amount (i.e. concentration) of the analyte. In somecases, optional adjustments may be made to accommodate factors such asthe natural occurrence of the isotope or impurity of the added isotope.The reference standard preferably interacts with a SERS substrate (suchas a gold nanoparticle) similarly to the analyte of interest. Thus boththe reference standard and analyte show similar changes in signalstrength due to “hot spots” or variations in sample composition.Therefore, when the Raman spectrum for the analyte (i.e. the intensityof one or more of its characteristic bands) is scaled with reference tothe Raman spectrum for the reference standard (i.e. the intensity of oneor more of its characteristic bands), an accurate quantification isachieved.

In some cases, an isotopologue used as a reference standard may be madeby synthesizing a compound with, for example, a hydrogen isotope (i.e.deuterium) or an oxygen isotope (i.e. ¹⁸O). Replacing some or all of thehydrogen with deuterium causes a shift in the peaks of the Ramanspectrum for many compounds, including amines like monoethanolamine,methylamine, diethanolamine, cyclohexylamine, morpholine andmethyldiethanolamine, and hydrocarbons like benzene, toluene,ethylbenzene, and xylene. Replacing at least some, but preferably all,of the oxygen with heavy oxygen (typically ¹⁸O) causes a shift in thepeaks of the Raman spectrum for many molecular ions, perchlorate,sulfate and selenate ions, for example.

Some analytes may be inherently SERS active—i.e., they interactnaturally with a SERS substrate to produce a strong signal. In othercases, analytes may be made to interact with a SERS substrate by theaddition of a binding reagent or adjustment of the general sampleconditions such as pH or ionic strength. In yet other cases, a reactioninvolving the analyte is used to produce or consume a more SERS activecompound. The more SERS active compound could be a more inherentlyactive compound or a compound that can be more easily made to interactwith a SERS substrate by way of a binding agent. The amount of the moreSERS active compound after the reaction can be measured and compared toa known amount of the more SERS active compound, if any, that waspresent before the reaction started. An internal standard for the moreSERS active compound can be used to increase the accuracy of themeasurement. The amount of the more SERS active compound that isconsumed or created in the reaction can then be used to calculate theconcentration of the analyte using a formula for the reaction.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic drawing of a portable SERS analysis system

FIG. 2 is a top view of a cartridge for use with the system of FIG. 1.

FIG. 3 shows an example optical system contained within the analysisinstrument according to the present invention.

FIG. 4 is a calibration curve for diethanolamine showing the accuracy,linearity, and detection range.

FIG. 5 is a calibration curve for perchlorate showing the accuracy,linearity, and detection range.

FIG. 6 is a calibration curve for selenate showing the accuracy,linearity, and detection range.

FIG. 7 shows a method for sample preparation in the analysis of aminesin sour water.

DETAILED DESCRIPTION

An exemplary water analysis system described herein can be used toprovide on-site or in-field analysis of aqueous samples. In general, atechnician collects a water sample, processes that sample for analysis,and then introduces the sample to a cartridge. The cartridge is insertedinto a Raman spectroscopy device, which may be operated through aninterface provided by a general-purpose computer. Optics and electronicswithin the Raman spectroscopy device produce a Raman spectrum for thesample which is analyzed, for example with software in thegeneral-purpose computer, to determine the concentration of an analyteunder investigation in the water sample.

Raman spectroscopy allows analytes to be detected by their specific“fingerprint”, or pattern of peaks in the Raman spectrum. Preferably, aknown amount of a reference compound is present in the sample with theanalyte when the Raman spectrum is produced and provides an internalcalibration standard against which signal intensity of the analyte maybe compared to provide accurate quantitative results. The intensity of aRaman signal scales with (i.e. is proportional to) the number ofmolecules present per unit area. When a known amount of calibrationstandard is introduced to a sample aqueous solution of known volumecontaining the analyte, the quantity of analyte may be determined bycomparing the signal intensity of the calibration standard and theanalyte, for example through ratiometric methods.

The specific ratiometric method used is not critical. For example, theconcentration of the reference compound in the sample can be divided bythe intensity of the highest peak in the spectrum for the referencecompound to provide a correlation factor. Multiplying this correlationfactor by the intensity of the highest peak in the spectrum for theanalyte produces the concentration of the analyte. Alternatively, thesignal intensity of the analyte (i.e. the intensity within one or morebands characteristic of the analyte) can be divided by the signalintensity within corresponding bands of the reference compound toprovide a scaling factor. Multiplying this scaling factor by the knownconcentration of the reference compound in the sample produces theconcentration of the analyte. Optionally adjustments may be made forvarious factors such as the purity of the calibration standard, ornatural occurrence of the calibration standard in the analyte. Otherordinary adjustments, such as smoothing the spectrum curve orsubtracting background signals before measuring intensity, may also bemade.

The reference compound may be added to the cartridge duringmanufacturing or added to a sample containing the analyte before theanalyte is added to the cartridge. Similarly, SERS active metallicnanoparticles (i.e. markers) may be added to the cartridge duringmanufacturing or added to a sample containing the analyte before theanalyte is added to the cartridge. Optionally, mixing a solution of thereference compound with an aqueous sample containing the analyte as afirst step, or at least before loading the sample to the cartridge,allows a mixture of analyte and reference compound to be made usingvolumes larger than would fit in the cartridge. This can help withmaking an accurate calculation of the concentration of the referencecompound in the mixture, particularly when making measurements in thefield where high precision liquid handling devices are not available.Once the reference compound and analyte sample are mixed together, latererrors such as spilling some of the mixture do not typically affect theaccuracy of the analyte concentration measurement.

The reference compound is added to the mixture in an amount similar tothe expected amount of the analyte. For example, if the analyte isexpected to have a concentration in the range of 0-100 ppm, the analytesample may be mixed with a solution of equal volume containing 25-75 ppmof reference compound. Similarly, if the analyte is expected to have aconcentration in the range of 0-100 ppb, the analyte sample may be mixedwith a solution of equal volume containing 25-75 ppb of referencecompound. The amount of reference compound in the mixture is preferablywithin 0.1 to 10 times the amount of anylate in the mixture.

Optionally, the reference compound may be an isotopologue of the analyteunder investigation. An isotopologue provides a powerful internalcalibration standard as it differs from the analyte only in the numberof neutrons. The chemical response and reaction of the referencecompound will be nearly identical to the analyte. However, under Ramanspectroscopy, the isotopologue has a different spectrum. Therefore, onecan record the analyte and isotopologue spectra simultaneously, usingratiometric analysis of the composite spectrum to quantify the unknownanalyte.

In another option, the calibration standard may be an isotope of acompound that is chemically similar to the analyte under study. Forexample, selenium and sulfur are chemically quite similar. A measurementof selenate may rely upon the measurement of sulfate, of a selenateisotope, or of a sulfate isotope as a calibration standard. In caseswhere an unknown amount of sulfate may exist in the sample, for examplewhen testing flue gas desulfurization blowdown water, the use of aselenate isotope or sulfate isotope is preferred. Sulfate isotopes aretypically easier to produce. When either a selenate or sulfate isotopeis used, the concentration of sulfate in the sample can be measuredsimultaneously with a measurement of the selenate concentration.

Optionally, one or more additional compounds may be mixed with thesample to enhance interaction between the nanoparticles and both of theanalyte and the reference compound. In some cases, the additionalcompound modifies the SERS marker, i.e. the nanoparticles. Optionally,the modification can be chosen for analyte specificity using compoundsdesigned to interact preferentially or only with the analyte underinvestigation and the reference compound. This approach reducesinterferences while increasing signal strength.

Optionally, an additional compound can include one or more compoundsselected from: thiols, amines, silanes, polymeric particles, metallicparticles, crown esters, cysteamine, cystamine, diethylaminethanethiol,mercaptopropionic acid, 1-propanethiol, octanethiol, octyldecanethiol,polystyrene, iron, or silica. In other options, the additional compoundcan be a compound effective to modify the pH or ionic strength of amixture including the analyte and the nanoparticles. Some analytesinteract with a SERS substrate more at a certain pH or in the presenceof ions. Optionally, one or more additional compounds can be providedpremixed with the reference compound in a reagent solution.

FIG. 1 shows an analysis system 10. The analysis system 10 includes aRaman spectroscopy unit 12, a computer 14 and a cartridge 16. Thecomputer 14 may be a separate computer as shown or alternatively couldbe incorporated into the Raman spectroscopy unit 12. The computer 14 ispreferably a portable general-purpose computer, for example a laptop,tablet or smart-phone.

The Raman spectroscopy unit 12 includes an optical detection system in ahousing. The optical detection system is capable of providing anilluminating electric field, where the illuminating electric field iscapable of being used for Raman spectroscopy, for example withRaman-scattering nanoparticles and the calibration solution, to analyzea sample under test input on or the cartridge 16. The Raman spectroscopyunit 12 further includes a lens 20 with a focus ring 18. The focus ring18 allows the lens 20 to be focused on a sample contained in thecartridge 16. The Raman spectroscopy unit 12 includes a cartridge holder22 adapted to receive the cartridge 16. The cartridge holder 22 can betranslated relative to lens 20 by turning a screw 24. A second screw(not shown) is preferably provided orthogonal to screw 24 to allow aportion of the cartridge 16 at which a sample to be tested is visible tobe aligned with the lens 20.

The cartridge 16 receives an aqueous mixture including the analyte. Thecartridge 16 has a cavity 26, which receives the sample. The cartridge16 may be plastic. However, if the mixture is expected to be transparentsuch that the Raman laser would penetrate through to the bottom of thecavity 26, the bottom of the cavity may be lined with a reflectivesurface such as stainless steel. The cartridge 16 may alternatively be aglass or plastic bag or vial or other suitable container. When thecartridge 16 is mounted in the cartridge holder 22, the cavity 26 isaligned with the lens 20.

In another option, the cartridge 16 can include one or more ofnanoparticles, a reference compound, one or more additional compounds,microfluidic channels configured to provide an area with an increaseddensity of nanoparticles, or microfluidic separation channels, forexample as described in U.S. Pat. No. 8,070,956.

FIG. 3 shows an example optical system 1400, contained within the Ramanspectroscopy unit 12. Shown is a light source 1402, such as a laser,projecting a light beam 1406 passing through a series of optics 1408arranged as a beam expander that is reflected into a dichroic optic 1410to direct the reference light beam 1411 into a spectrometer 1412 foranalysis in a monochrometer 1414 and recordation in a CCD array 1416.The dichroic 1410 simultaneously directs the signal light beam 1413 tothe cartridge 1418 to gather a signal from a sample in the cartridge1418 and reflect the signal along the beam path into the spectrometer1412 and CCD 1416 array for analysis.

Quantification of Amines

One important analyte category is the monitoring of amines, for examplein industrial processes. Organic amines are used as corrosion controlagents that increase pH and scavenge corrosive contaminants.Monoethanolamine (MEA), for example, is a widely used corrosioninhibitor that reduces dissolved CO₂ and helps control pH in industrialboilers and nuclear power plants. Amines are also effective as hydrogensulfide scavengers in oil and gas production and processing. Forexample, MEA-triazine is frequently used as a hydrogen sulfide scavengerat the well-head. MEA or other compounds can also exist as tramp aminesthat affect refinery operations. On-site monitoring for amines, forexample at a refinery, pipeline, or well-head, can help maintainappropriate corrosion protection while extending system lifetime andavoiding costly corrosion-induced shutdowns and failures.

The analysis system 10 described above may be operated to measure theconcentration of an amine. Amines interact naturally with gold andsilver substrates. Under basic pH conditions, the amine group willadhere to the nanoparticles, resulting in a strong Raman response. Insome cases, the analysis method takes under five minutes, and may beperformed in the field. In some cases, measurement of an amine in theppb range is possible. However, in industrial process management,measurement of amines in the ppm range is more often required. Ingeneral, the concentration of an amine can be measured using anisotopologue of an amine as a reference compound, preferably anisotopologue of the same amine or an amine of similar molecular weightand structure. An anylate, isotopologue and nanoparticles mixture ispreferably adjusted to a basic pH, for example a pH of at least 1 orpreferably at least 2 higher than the pKa of the anylate amine, beforemeasuring its Raman spectrum.

In one embodiment, a method provides part-per-million concentrationmeasurement of methylamine in refinery process waters. Methylamine is atramp amine that negatively impacts the refining process. The detectionlimit for methylamine in aqueous solutions is 20-ppb or better.

In another embodiment, the concentration of monoethanolamine (MEA) isdetermined. Rapid measurement of monoethanolamine is useful incontrolling refinery operations. A preferred isotopologue for MEA ismonoethanolamine-d4. pH of the MEA, isotopologue and nanoparticlesmixture is preferably raised to 12.6 to 12.9. The mixture of MEA,isotopologue and nanoparticles is added to the cartridge and its Ramanspectrum determined.

In a method described herein, a reference compound is added to an aminecompound analyte sample. The reference compound is an isotopologue ofthe same or another amine compound. In one example, a known volume ofmethylamine-d₃ solution at 25- to 75-ppm is added to a sample of knownvolume (preferably the same or within 50% of the volume ofmethylamine-d₃ solution) having an unknown quantity of methylamine-d₀.The methylamine-d₃ creates a reference spectrum (within a compositespectrum) against which the methylamine-d₀ spectrum may be quantified.This approach can result in ±10% measurement accuracy over a 0- to100-ppm range of methylamine-d₀. Accuracy can be improved at the low endwith a lower concentration methylamine-d₃ solution. pH of the mixture ispreferably adjusted to 12.6 to 13.0 before taking its Raman spectrum.

In another example, diethanolamine-d₈ is used as an internal standardwhen measuring the concentration of diethanolamine. FIG. 4 shows acalibration curve prepared by measuring the concentration of samples ofdiethanolamine prepared at different concentrations withdiethanolamine-d₈ used as an internal standard. The response is linearover 0- to 100-ppm, and the accuracy of the measured concentration iswithin 10%. pH of the mixture is preferably adjusted to 12.6 to 13.0.

In other examples, the approach described above is applied totriazine-based compounds. For example, accurate, ppm-level measurementsof MEA-triazine or dithiazine can be achieved using an ethanolamine-d₄or an MEA-triazine-d₁₂ isotopologue as a reference compound.

In further examples, the process for measuring one amine can be extendedto measuring multiple amines. The internal standard can be a mixture ofmaterials, for example ethanolamine-d₄ and methylamine-d₃. These twocompounds individually are excellent internal standards for ethanolamineand methylamine individually. By including both isotopologues in amixture, one can determine the concentration of ethanolamine andmethylamine in a sample. First, the spectrum is scaled to theethanolamine-d₄ peak at 870-cm⁻¹, and the ethanolamine concentration isdetermined. The same spectrum is then scaled to the 950-cm⁻¹methylamine-d₃ peak, and the methylamine concentration is determined.Furthermore, the spectrum for a first amine may be subtracted from theoverall spectrum sequentially before analyzing the spectrum of a secondamine to improve signal-to-noise ratio. For example, an ethanolaminespectrum is stronger than a methylamine signal. By subtracting theethanolamine spectrum, one can improve the accuracy of methylamineanalysis.

Use of an Intermediate Reaction

In yet other examples, a reaction involving the analyte is used toproduce or consume a more SERS active compound. The amount of the moreSERS active compound in the reaction product is measured to determine ifSERS active compound was created or consumed. A reference compound forthe more SERS active compound can be used to increase the accuracy ofthe measurement. The amount of the more SERS active compound that isconsumed or created in the reaction can then be used to calculate theconcentration of the analyte using a formula for the reaction.

In one example, the consumption of pyridine is used to determine theconcentration of a gem-halogenated compound. The amount ofgem-halogenated compound required to consume a certain amount ofpyridine can be determined according the Fujiwara reaction, normallyused in a colorimetric method for the detection of gem-halogenatedcompounds using pyridine. The gem-halogenated compound may be, forexample, trichloroethylene, a trihalomethane, chloroform, or ahaloacetic acid

In one example, the concentration of a gem-halogenated compound, inparticular trichloroethylene (TCE), is measured. A sample is taken ofwater suspected of containing TCE in the ppb range. The pH of the sampleis adjusted to 12-13, for example by adding caustic, as needed for theFujiwara reaction. Dilute pyridine solution, for example at about100-150 ppb, is added in a 1:1 or similar volume ratio to the alkalinesample. The reaction of pyridine with TCE will progress, resulting incomplete consumption of the TCE and a decrease in the pyridineconcentration. We then add an internal standard, for example a solutionof pyridine-d₅ of known concentration and volume, to the reactionproduct solution. When mixed with a SERS substrate such as goldnanoparticles, the resulting SERS signal will be a combination ofpyridine-d₀ and pyridine-d₅ signals. We then scale the signal to themain pyridine-d₅ peak, which is spectrally shifted from the main d₀peak, to determine the amount of pyridine-d₀ remaining. Subtracting thisvalue from the initial amount of pyridine gives the amount of pyridineconsumed. This value can be used to calculate the amount of TCE that waspresent in the original sample. If there is no pyridine left afterreaction then the test is inconclusive and must be repeated with alarger initial amount of pyridine.

Alternatively, a pyridine derivative such as nicotinamide may be used todetermine the concentration of a gem-halogenated compound. Nicotinamidemay offer benefits such as selectivity, usability, or safety. Pyrdine-d₅may be used as an internal standard with a pyridine derivative, as mayan isotopologue of the pyridine derivative.

In another example, the concentration of formaldehyde is measured by wayof a reaction between an amine and formaldehyde. Cysteamine reacts withformaldehyde to create a thiazolidine. Cysteamine is also a thiol thatinteracts strongly with a gold substrate. A sample of formaldehyde ismixed with a known amount of cysteamine. The reaction completelyconsumes the formaldehyde and some of the cysteamine. This reactedproduct solution may then be mixed with a cysteamine isotopologue andgold nanoparticles. The ratio of cysteamine isotopologue to cysteamineprovides a measure of cysteamine remaining, which can be used todetermine the initial formaldehyde concentration. Alternatively, athiazolidine isotopologue can be added to the reacted product solutionto provide a measure of cysteamine remaining, which can be used todetermine the initial formaldehyde concentration. In either case, thelack of any remaining cysteamine indicates a failure and that the methodshould be repeated starting with a larger amount of cysteamine. In theseexamples, cystamine may be used in place of cysteamine.

Quantification of Ions

The methods and equipment described above may also be adopted to measurethe concentration of ions, for example molecular anions, in water. Theconcentration may be in the ppb range. In addition to a referencecompound, a binding compound is used to increase interaction between theion and the nanoparticles. The binding compound may be an amine,preferably with no more than 3 carbon atoms between a nitrogen atom anda sulfur atom, for example a thiol. A mixture of anylate, isotopologue,binding compound and nanoparticles is adjusted to an acidic pH, forexample 5.0 or less. The preferred pH can be determined by trial anderror, but varies with the pKa of the binding compound and can beestimated from the examples below adjusted to reflect the pKa of adifferent amine if used.

In one example, the concentration of perchlorate ions is measured.Perchlorate is a hydrophobic anion. By first modifying the surface ofthe SERS nanoparticles, i.e. gold nanoparticles, with a hydrophobiccationic species, we create a surface interaction between perchlorateand the nanoparticle. Under acidic conditions, amines become protonated,acting as a cationic treatment to draw anionic species to thenanoparticle. Thiol-based amine compounds like dimethylaminoethanethiolor diethylaminoethanethiol create such a surface. Other anions likesulfate or selenate that are not hydrophobic interact with SERSnanoparticles, i.e. gold nanoparticles, under the influence of lesshydrophobic compounds such as cysteamine. The addition of an anionicisotopologue as an internal standard enables repeatable, accuratemeasurements.

In a particular example of perchlorate measurement, the user starts withat 100-μl sample of water expected to contain perchlorate. To thissample, 10-μl of a 550-ppb perchlorate-¹⁸O₄ solution is added, resultingin a final concentration of 50-ppb Cl¹⁸O₄ in the sample. This sample isthen adjusted to pH 1.8-3.0, preferably about 2.5 using hydrochloricacid. Next, a small quantity of methanol (10-μl) and a preferred thiolcompound (dimethylaminoethanethiol) is added to the sample vial,followed by 5-ul of concentrated gold nanoparticles. This combinedsample is allowed to dry on a clean, steel substrate, and then analyzedvia Raman spectroscopy. The heavy perchlorate provides an internalstandard against which the intensity of the perchlorate spectrum iscompared. The resulting calibration curve is presented in FIG. 5.Measurement accuracies of 10% with 4-ppb detection limits are possible.A similar approach works for the detection and quantification ofchromate, using the heavy-oxygen isotopologue of chromate as an internalstandard.

In cases where creation of the isotopologue would be unduly expensive orcomplicated, a similar anion can be used as a reference material. Asimilar anion preferably has the same number and kind of electronacceptors attached to an electron donor with the same type of bonds. Forexample, selenium and sulfur have very similar structures. The detectionof selenate may be achieved by using sulfate or a selenate isotopologue(preferably with 4 ¹⁸O) as an internal standard, with cysteamine orcystamine as a nanoparticle surface treatment compound. The pH of themixture of selenate, isotopologue, cysteamine or cystamine and SERSsubstrate (for example gold nanoparticles) is preferably adjust to about3.5, for example by adding HCl. However, sulfate is a commonly occurringmaterial, making a sulfate isotopologue (S¹⁸O₄) a convenient internalstandard. Furthermore, by using a sulfate isotopologue, both sulfate andselenate can by quantified simultaneously. A calibration curve ofselenate measured using a sulfate isotopologue as an internal standardis presented in FIG. 6.

In preparing an isotopologue of a molecular anion, it is preferably toreplace all of the O atoms with ¹⁸O. This requires a reaction driven tocompletion, which more easily creates a substantially pure isotopologuethan a partial substitution of oxygen ions. The natural occurrence ofsuch isotopologues is also rare and so adjustments are generally notrequired for isotopologue purity or naturally occurring isotopologues.However, the isotopologue should not be exposed to conditions that wouldcause it to revert to the naturally occurring form. For example, aselenate isotopologue with ¹⁸O should not be stored in highly acidicwater (i.e. pH of 1.0 or less) for an extended period of time since thiswill cause it to swap its heavy oxygen with oxygen in the water.

Although the nanoparticles may be pre-treated with an additionalcompound such as a thiol before adding the sample, it is preferable thatthe additional compound be mixed with the nanoparticles at about thesame time the sample. This process increases response while improvingproduct lifetime.

In other examples, addition of anionic compound, such as3-mercaptopropionic acid, to nanoparticles enable the measurement ofcationic ions in water. As discussed above, an internal referencestandard for the cationic ions is preferably also added.

Quantification in Complex Matrices

In some cases, a measurement method as described above may be applied toanalyte measurement in complex sample matrices. For example, refineryprocess waters may contain ppm-level amines in the presence of manysalts and hydrocarbons. Pre-treatment methods can prepare the sample foranalysis; however, pre-treatment can be challenging as it may affect thelevel of the analyte of interest. In these cases, adding the referencecompound before treatment reduces errors caused by pre-treatment. Thepre-treatment may be, for example, solid-liquid separation, ionexchange, strong anion exchange or ion extraction.

For example, the measurement of amines in refinery sour water presents asignificant challenge. Sour water is defined by the presence of sulfide,which leads to the distinctive odor. These water samples, however, canalso contain an array of other contaminants: ionic species, metals,organic acids, hydrocarbons, and amines, to name a few. Water collectedfrom desalter operations, for example, may have high ion and amineslevels (potentially more than 1000-ppm). Overhead water, in contrast,typically has contaminants levels below 100-ppm.

To analyze complex samples, the user first introduces the internalstandard, such as an isotopologue, to the sample before samplepre-treatment. The sample pre-treatment will remove the analyte and theinternal standard generally equally, enabling a precise concentrationmeasurement after the pre-treatment. The pre-treatment is preferably aprocess to remove anions, for example an anion exchange or anionextraction process. The pH of the mixture of analyte and isotopologue ispreferably reduced as required to keep the analyte and isotopologue insolution while undesirable anions are removed in the pretreatmentprocess. Alternatively, the analyte and isotopologue maybe captured bypassing the mixture through a cation extraction unit, discarding theremainder of the sample, and then eluting the analyte and isotopologuefrom the cation extraction unit.

For sour water analysis, an exemplary sample preparation method ispresented in FIG. 7. A solid-phase extraction (SPE) column designed forthe removal of anions is first cleaned with 5-mL of 0.01 M HCl. The500-μL sample is mixed 1:1 with a reagent solution containing theisotopologue. 10 μL of 1 M HCl is added to this mixture and then themixture is introduced to the SPE column. The first 500-μL is discarded,and the remaining sample is collected. The pH of the remaining sample isadjusted upwards and then its Raman spectrum is measured.

U.S. application Ser. No. 14/198,163 filed on Mar. 5, 2014 isincorporated herein by reference.

The present invention has now been described in accordance with severalexemplary embodiments, which are intended to be illustrative in allaspects, rather than restrictive. Thus, the present invention is capableof many variations in detailed implementation, which may be derived fromthe description contained herein by a person of ordinary skill in theart. For example, many additional amines may be detected using a similarapproach. These amines include methylamine, diethanolamine,methyldiethanolamine, dimethylethanolamine, diisopropylamine,cyclohexylamine, morpholine, and methoxypropylamine. Other anionsincluding nitrate, chromate, thiosulfate, phosphate, and carbonate maybe detected using similar approaches to the method presented forperchlorate analysis.

All such variations are considered to be within the scope and spirit ofthe present invention as defined by the following claims and their legalequivalents.

We claim:
 1. A method of measuring the amount of an analyte in water, the method comprising the steps of: obtaining a sample of water of known volume containing an analyte; mixing a known amount of an isotopologue with the sample; mixing Raman-scattering nanoparticles with the sample; producing a Raman spectrum of the mixture; and, performing a ratiometric analysis of peaks or bands within the Raman spectrum corresponding to the analyte and the isotopologue within the Raman spectrum to quantify the analyte, wherein the analyte is perchlorate and the isotopologue is a heavy-oxygen isotopologue of perchlorate, further comprising adding a thiol-based amine compound to the mixture and adjusting the pH of the mixture to 1.8-3.0.
 2. The method of claim 1 wherein the heavy-oxygen isotopologue of perchlorate has all oxygen atoms substituted with ¹⁸O.
 3. The method of claim 1 wherein the thiol-based amine compound is dimethylaminoethanethiol or diethylaminoethanethiol.
 4. The method of claim 3 further comprising adding methanol to the mixture.
 5. A method of measuring the amount of an analyte in water, the method comprising the steps of: obtaining a sample of water of known volume containing an analyte; mixing a known amount of an isotopologue with the sample; mixing Raman-scattering nanoparticles with the sample; producing a Raman spectrum of the mixture; performing a ratiometric analysis of peaks or bands within the Raman spectrum corresponding to the analyte and the isotopoloque within the Raman spectrum to quantify the analyte; and, pre-treating the mixture after adding the isotopoloque but before producing the Raman spectrum, wherein the pre-treating step comprises removing anions from the mixture.
 6. The method of claim 5 wherein the analyte is an amine and the isotopologue is an amine with one or more hydrogen atoms replaced with deuterium.
 7. The method of claim 6 comprising adjusting the pH of the mixture to a pH at least one more, or at least 2 more, than the pKa of the analyte.
 8. The method of claim 6 wherein the analyte is MEA and the isotopologue is an MEA isotopologue.
 9. The method of claim 6 wherein the analyte is methylamine and the isotopologue is methylamine-d₃.
 10. The method of claim 6 wherein the analyte is diethanolamine and the isotopologue is diethanolamine-d₈.
 11. The method of claim 6 wherein the analytes are ethanolamine and methylamine and the isotopolgues are ethanolamine-d₄ and methylamine-d₃.
 12. The method of claim 5 wherein said is Raman-scattering nanoparticles are comprised of gold or silver particles.
 13. The method of claim 12 wherein said nanoparticles are 10 to 200 nm in diameter.
 14. The method of claim 5 wherein the water is refinery sour water and the pre-treating step comprises reducing the pH of the mixture before removing anions from the mixture and increasing the pH of the mixture before producing the Raman spectrum of the mixture.
 15. A method of measuring the amount of an analyte in water, the method comprising the steps of: obtaining a sample of water of known volume containing an analyte; reacting the analyte with a compound to consume the compound or produce a second compound; mixing a known amount of an isotopologue of the compound or the second compound with the reaction product; mixing Raman-scattering nanoparticles with the reaction product; producing a Raman spectrum of the mixture; and, performing a ratiometric analysis of peaks or bands within the Raman spectrum corresponding to the compound or second compound or both and the isotopologue within the Raman spectrum to quantify the analyte.
 16. The method of claim 15 wherein the compound or second compound is an amine and further comprising adding an isotopologue of an amine to the mixture.
 17. The method of claim 15 wherein the analyte is formaldehyde, the compound is an amine and the second compound is a thiazolidine.
 18. The method of claim 17 wherein the compound is cysteamine or cystamine.
 19. The method of claim 17 wherein the isotopologue is an isotopologue of the compound and the method comprises performing a ratiometric analysis of peaks or bands within the Raman spectrum corresponding to the compound and the isotopologue within the Raman spectrum to quantify the analyte.
 20. The method of claim 17 wherein the isotopologue is an isotopologue of the second compound and the method comprises performing a ratiometric analysis of peaks or bands within the Raman spectrum corresponding to the second compound and the isotopologue within the Raman spectrum to quantify the analyte. 